Project Summary: Neurodegenerative diseases remain some of the most difficult human maladies to understand, much less clinically care for. Fundamental research into basic neurobiology is needed to clarify disease mechanisms and provide research options for possible therapeutic care. Neurons are polarized cells in the nervous system that receive and transmit electrical and chemical information. Many cellular mechanisms support this neuronal function from cytoskeletal structure, nuclear gene expression, to polarized organelle trafficking. Mitochondria are organelles that support a variety of neuronal functions at specialized sites in axons and dendrites and maintain very different dynamics in each compartment. Mitochondrial morphology and trafficking behavior reflect mitochondrial function in a variety cell types and disruption of normal dynamics are related to cellular dysfunction and human disease. Moreover, dysfunctional mitochondria are a hallmark of neurodegenerative diseases. The study of mitochondrial morphology, dynamics, and trafficking in mammalian hippocampal neurons will pave the way forward to a better understanding of how mitochondrial networks are maintained in neurons. This study will focus on determining to what extent mitochondria are specialized in neuronal sub-compartments. Using advanced live cell fluorescent techniques, microfluidic platforms, and next generation proteomics and RNAseq analysis, we will identify the molecular mechanisms behind the formation of mitochondrial polarity and maintenance of mitochondrial networks in mature neurons. Furthermore, literature and our initial insights, support a role for mitofusin 2 in regulating neuronal mitochondrial networks. We are developing knock-OFF technology to study the role of Mfn2 as it pertains to mitochondrial fusion in neurons. Continued development of this new technology will provide a proof of concept and we believe will be generally applicable to the majority of membrane proteins. Transmembrane proteins present a special challenge when trying to acutely inactivate them, there is an urgent need for development of knock-OFF. Furthermore, individual mitochondria from different sub- compartments in neurons will be physically isolated through novel sorting and purification procedures. These mitochondria will be assayed for their proteome to gain insight into how mitochondrial sort and ultimately how the mitochondrial network is maintained. Insights from these studies will be used to understand the neuronal pathology resulting from an atypical congenital muscular dystrophy caused by mutations in choline kinase beta (CHKB).